We assessed the impact of the Janus Kinase (JAK) 1 inhibitor itacitinib on xenogeneic graft-versus-host disease (xGVHD). XGVHD was induced by i.v. injection 20 × 10⁶ human peripheral blood mononuclear cells (hPBMC) in NSG mice on day 0. Itacitinib (3 mg, ≈120 mg/kg) or methylcellulose was administered by force-feeding twice a day from day 3 to day 28. Mice were followed for xGVHD score and survival. In addition, human T-cell engraftment and as well as human T-cell subtypes were monitored in blood on days 14, 21, and 28 after transplantation. We observed that itacitinib-treated mice had significantly longer survival than control mice (median 45 versus 33 days; P < 0.001). Further, they also had lower absolute numbers of human CD4⁺ T cells on days 21 and 28 after transplantation as well as of human CD8⁺ T cells on days 14, 21, and 28 after transplantation. In addition, itacitinib-treated mice had higher frequencies of human regulatory T cells (Treg) on days 21 and 28 after transplantation. In summary, our data indicate that itacitinib decreases human T-cell engraftment, increases Treg frequencies and attenuates xGVHD in NSG mice transplanted with hPBMC.